• 한국균학회지
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• 제40권4호
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• pp.224-228
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• 2012

A. nidulans ${\alpha}$-COP과 상호작용하는 단백질을 동정하기 위하여 ${\alpha}$-COP을 암호하는 유전자를 bait로 yeast two-hybrid 스크리닝용 A. nidulans cDNA 라이브러리를 탐색한 결과, COPI 소낭의 구성요소 중 하나인 ${\varepsilon}$-COP을 암호화하고 있는 유전자를 동정하고 $aneA^+$($\underline{A}$spergillus $\underline{n}$idulans $\underline{e}$psilon-COP, $AN{\varepsilon}$-COP)으로 명명하였다. $aneA^+$ 유전자는 총 296개의 아미노산을 암호화하고 있으며, 다른 균류의 ${\varepsilon}$-COP과 높은 상동성을 보였다. Yeast two hybrid 시스템으로 두 단백질 간의 상호작용 부위를 분석한 결과, ${\alpha}$-COP의 COOH 도메인과 ${\varepsilon}$-COP의 C-말단부가 필수 부위였으며, ${\alpha}$-COP N-말단의 WD 도메인과 ${\varepsilon}$-COP의 TPR 부위는 두 단백질 간의 결합을 촉진하는 조절부위로 밝혀졌다. 또한 사상균인 A. nidulans와 효모류인 S. cerevisiae에서 ${\alpha}$-COP과 ${\varepsilon}$-COP 간 작용양상이 유사한 것으로 보아, COPI 소낭의 구성요소인 ${\alpha}$-COP과 ${\varepsilon}$-COP 간의 상호작용 기전은 진핵세포 내에서 진화적으로 잘 보존되어 있는 것으로 추정되었다.

• Mycobiology
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• 제43권1호
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• pp.31-36
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• 2015

We have previously isolated ${\varepsilon}$-COP, the ${\alpha}$-COP interactor in COPI of Aspergillus nidulans, by yeast two-hybrid screening. To understand the function of ${\varepsilon}$-COP, the $aneA^+$ gene for ${\varepsilon}$-COP/AneA was deleted by homologous recombination using a gene-specific disruption cassette. Deletion of the ${\varepsilon}$-COP gene showed no detectable changes in vegetative growth or asexual development, but resulted in decrease in the production of the fruiting body, cleistothecium, under conditions favorable for sexual development. Unlike in the budding yeast Saccharomyces cerevisiae, in A. nidulans, over-expression of ${\varepsilon}$-COP did not rescue the thermo-sensitive growth defect of the ${\alpha}$-COP mutant at $42^{\circ}C$. Together, these data show that ${\varepsilon}$-COP is not essential for viability, but it plays a role in fruiting body formation in A. nidulans.

• Molecules and Cells
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• 제38권10호
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• pp.866-875
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• 2015

COPI vesicles are essential to the retrograde transport of proteins in the early secretory pathway. The COPI coatomer complex consists of seven subunits, termed ${\alpha}-$, ${\beta}-$, ${\beta}^{\prime}-$, ${\gamma}-$, ${\delta}-$, ${\varepsilon}-$, and ${\zeta}$-COP, in yeast and mammals. Plant genomes have homologs of these subunits, but the essentiality of their cellular functions has hampered the functional characterization of the subunit genes in plants. Here we have employed virus-induced gene silencing (VIGS) and dexamethasone (DEX)-inducible RNAi of the COPI subunit genes to study the in vivo functions of the COPI coatomer complex in plants. The ${\beta}^{\prime}-$, ${\gamma}-$, and ${\delta}$-COP subunits localized to the Golgi as GFP-fusion proteins and interacted with each other in the Golgi. Silencing of ${\beta}^{\prime}-$, ${\gamma}-$, and ${\delta}$-COP by VIGS resulted in growth arrest and acute plant death in Nicotiana benthamiana, with the affected leaf cells exhibiting morphological markers of programmed cell death. Depletion of the COPI subunits resulted in disruption of the Golgi structure and accumulation of autolysosome-like structures in earlier stages of gene silencing. In tobacco BY-2 cells, DEX-inducible RNAi of ${\beta}^{\prime}$-COP caused aberrant cell plate formation during cytokinesis. Collectively, these results suggest that COPI vesicles are essential to plant growth and survival by maintaining the Golgi apparatus and modulating cell plate formation.